Deoxyribonucleoside monophospate bypass therapy for mitochondrial dna depletion syndrome

ABSTRACT

The invention relates generally to a pharmacological therapy for a human genetic diseases, specifically mitochondrial DNA depletion syndromes, and more specifically, thymidine kinase 2 (TK2) deficiency. The pharmacological therapy involves the administration of at least one deoxyribonucleoside monophosphate, or mixtures thereof. For the treatment of TK2 deficiency, the pharmacological therapy involves the administration of either deoxythymidine monophosphate (dTMP) or deoxycytidine monophosphate (dCMP), or mixtures thereof. This molecular bypass approach is applicable to other disorders of unbalanced nucleotide pools, especially those found in mitochondrial DNA depletion syndrome.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. non-provisional patentapplication Ser. No. 15/082,207, filed Mar. 28, 2016, which claimspriority to U.S. provisional patent application Ser. No. 62/138,583filed Mar. 26, 2015, all of which are incorporated by reference, as ifexpressly set forth in their respective entireties herein.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under grant P01HD080642awarded by the NIH. The Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention relates generally to a pharmacological therapy for a humangenetic diseases, specifically mitochondrial DNA depletion syndromes,and more specifically, thymidine kinase 2 (TK2) deficiency. Thepharmacological therapy involves the administration of at least onedeoxyribonucleoside monophosphate, or mixtures thereof. For thetreatment of TK2 deficiency, the pharmacological therapy involves theadministration of either deoxythymidine monophosphate (dTMP) ordeoxycytidine monophosphate (dCMP), or mixtures thereof. This molecularbypass approach is applicable to other disorders of unbalancednucleotide pools, especially those found in mitochondrial DNA depletionsyndrome.

BACKGROUND OF THE INVENTION

Mitochondrial diseases are clinically heterogeneous diseases due todefects of the mitochondrial respiratory chain and oxidativephosphorylation, the biochemical pathways that converts energy inelectrons into adenosine triphosphate (ATP). The respiratory chain iscomprised of four multi-subunit enzymes (complexes I-IV) that transferelectrons to generate a proton gradient across the inner membrane ofmitochondria and the flow of protons through complex V drives ATPsynthesis (DiMauro and Schon 2003; DiMauro and. Hirano 2005). CoenzymeQ10 (CoQ10) is an essential molecule that shuttles electrons fromcomplexes I and II to complex III. The respiratory chain is unique ineukaryotic, e.g., mammalian, cells by virtue of being controlled by twogenomes, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). As aconsequence, mutations in either genome can cause mitochondrialdiseases. Most mitochondrial diseases affect multiple body organs andare typically fatal in childhood or early adult life. There are noproven effective treatments for mitochondrial diseases. CoQ10 and itsanalogs have been administered to patients with mitochondrial disease toenhance respiratory chain activity and to detoxify reactive oxygenspecies (ROS) that are toxic by-products of dysfunctional respiratorychain enzymes.

An important subgroup of mitochondrial diseases is mitochondrial DNAdepletion syndrome (MDS), which encompasses clinically and geneticallyheterogeneous disorders with reduction of mitochondrial DNA (mtDNA) copynumber in tissues, leading to insufficient synthesis of mitochondrialrespiratory chain complexes (RC) (Hirano et al., 2001). Mutations inseveral nuclear genes have been identified as causes of infantile MDS,including TK2, DGUOK, POLG, POLG2, SLC25A4, MPV17, RRM2B, SUCLA2,SUCLG1, TYMP, OPA1, and C10orf2 (PEO1) (Bourdon, et al. 2007; Copeland2008; Elpeleg, et al. 2005; Mandel, et al. 2001; Naviaux and Nguyen2004; Ostergaard, et al. 2007; Saada, et al. 2003; Sarzi, et al. 2007;Spinazzola, et al, 2006).

One of these genes is TK2 which encodes thymidine kinase (TK2), amitochondrial enzyme required for the phosphorylation of the pyrimidinenucleosides (deoxythymidine and deoxycytidine) to generatedeoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate(dCMP) (Saada, et al. 2001). Thus, mutations in TK2 impair themitochondrial nucleoside/nucleotide salvage pathways required forsynthesis of deoxynucleotide triphosphate (dNTP), the building blocksfor mtDNA replication and repair.

TK2 deficiency was first described in 2001 by Saada and colleagues(Saada, et al. 2001), in four affected children originating from fourdifferent families, who suffered from severe, devastating myopathy.After an uneventful early development, at ages 6-36 months the patientsdeveloped hyperCKemia, severe muscle hypotonia with subsequent loss ofspontaneous activity. Depletion of mtDNA was identified in muscle tissuewith 16-22% of residual mtDNA. The patients harbored recessive TK2mutations causing severe reductions in TK2 activity. As a consequence ofmtDNA depletion, enzymatic activities of complexes I, III, IV and V ofthe mitochondrial respiratory chain in muscle were significantlyreduced, whereas the activity of complex II, the only complex that doesnot contain mtDNA-encoded proteins, was relatively normal. The diseasewas rapidly progressive and two patients were mechanically ventilated at3 year, while two other patients were already dead by the time of thereport.

After the first description, sixty additional patients have beenreported in literature and at least twenty-six further patients havebeen diagnosed but not reported (Alston, et al. 2013; Bartesaghi, et al.2010; Bain, et al. 2012; Blakely, et al. 2008; Carrozzo, et al. 2003;Chanprasert, et al. 2013; Collins, et al. 2009; Galhiati, et al. 2006;Gotz, et al. 2008; Leshinsky-Silver, et al. 2008; Lesko, et al. 2010;Mancuso, et al. 2002; Mancuso, et al. 2003; Marti, et al. 2010; Oskoui,et al. 2006; Paradas, et al. 2012; Roos, et at. 2014; Tulinius, et al.2005; Tyynismaa, et al. 2012; Vila, et al. 2003; Wang, el al. 2005). Allninety patients had proximal muscle weakness with mild to severerespiratory insufficiency and an increased creatine kinase level up to20-fold above normal. Disease onset ranged from birth to 74 years ofage, but the majority of patients had infantile (less than 1 year,34.4%) or childhood onset (1-12 years, 46.6%) weakness. Adult-onset (18years or older) was reported in 14.4% of patients while only 2.2% showedfirst disease symptoms in adolescence (12-17 years). Global motorfunction was severely impaired in 47/58 (81% with Karnofsky or LanskyPerformance Status <50): 46 had motor regression and werewheelchair-bound at the last follow-up while one never acquired theability to walk. Twelve patients (3 children, 9 adults; 19%) had motorfunction compatible with nearly normal daily life at the last follow-up.They were able to walk independently, but required support for longdistance, climbing stairs, or both. Data from motor rating scale werenot available for 31 patients. Respiratory muscles were severelycompromised in 30/48 (62.5%) patients, who required mechanicalventilation or nocturnal/continuous non-invasive ventilation (data werenot available in 42 patients). Other muscular functions were affected in31/83 (37.3%) patients who manifested a variable combinations of:dysarthria/dysphasia (3); rigid spine (1); mild dysphagia (9); facialdiplegia (19); ptosis (22); and progressive external ophthalmoparesis(PEO) (12). Sixteen patients required gastrostomy tube because of severedysphagia and failure to thrive. Central nervous system was affected in13 out of 90 (14.5%) causing: recurrent seizures (6); encephalopathy(5); cognitive impairment (4); coma episodes (1); and sensorineuralhearing loss (3). TK2 deficiency caused death in the first 3 years oflife in 50% of the patients; 24/41 (58.5%) had infantile-onset while7/41 (17%) had a childhood onset. Only 6.25% of patients have lived morethan 42 years.

Nerve conduction studies (NCSs) and electromyography (EMG), performed in40 patients, showed: myopathic changes defined by polyphasic shortduration low amplitude motor units potentials in 32/40 (80%); “myopathicand neuropathic” changes in 3/40 (7.5%); sensory axonal neuropathy byNCS in one (2.5%); chronic denervation by EMG in another patient (2.5%);low amplitude in the facial nerve in one patient (2.5%); isolated“neuropathic” changes, in 2/40 (5%); and no abnormalities in threepatients (7.5%). Muscle biopsies revealed variable depletion andmultiple deletions of mitochondrial DNA (mtDNA).

Based on clinical and molecular genetics findings, three diseasepresentations were identified: i) infantile myopathy (37.8%) with onsetof weakness in the first year of life with severe mtDNA depletion andearly mortality; ii) childhood SMA-like myopathy (35.6%) with severemtDNA depletion; and iii) adult myopathy (26.7%) with mild weakness atonset and slow progression to loss of ambulation, respiratoryinsufficiency, or both, often with chronic progressive externalophthalmoparesis in adulthood in association with mtDNA multipledeletions, reduced mtDNA copy number, or both. Thus, TK2 deficiencymanifests a wide clinical and molecular genetic spectrum with themajority of patients manifesting in early childhood with a devastatingclinical course, while others have slowly progressive weakness overdecades.

Histological and histochernical study showed type I fiber prevalence,atrophic fibers with lipid storage and increased connective tissue,ragged red fibers, cytochrome c oxidase (COX, complex IV) negativefibers with succinate dehydrogenase (SDH, complex II) hyperactivity. Inthe end stage, muscle is replaced by fatty tissue, including therespiratory muscle, as evident in the chest MRI of one patient (Collins,et at. 2009).

Recently, adult cases have been reported, four with slowly progressivemyopathy and two with PEO. In these patients the levels of mtDNAdepletion was not severe and was associated with multiple deletions ofmtDNA (Béhin, et al. 2012; Paradas, et al. 2012; Tyynismaa, et al.2012).

Treatment for TK2 deficiency, like most MDS and mitochondrial disorders,has been limited to supportive therapies. Thus, there is a need forbetter therapeutic intervention, and understanding the pathomechanism ofMDS would allow the design of treatment strategies targeting either thecause of the disease or the downstream metabolic defects, making formore effective therapies.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method oftreating a disorder characterized by unbalanced nucleotide pools,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a composition comprising one or moredeoxyribonucleoside monophosphates.

In a preferred embodiment, the disorder is a mitochondrial DNA depletionsyndrome, and in a more preferred embodiment, the disorder is athymidine kinase 2 (TK2) deficiency.

Other MDS disorders that can be treated with the method of the currentinvention include but are not limited to, deficiencies in the: DGUOKgene, encoding deoxyguanosine kinase, dGK; RRM2B gene, encoding p53R2,the p53 inducible small subunit of ribonucleotide reductase, RNR; andTYMP gene, encoding thymidine phosphorylase, TP.

In a preferred embodiment, the deoxyribonucleoside monophosphates areeither deoxythymidine monophosphate (dTMP) or deoxycytidinemonophosphate (dCMP) or mixtures thereof. Deoxyadenosine monophosphate(dAMP) and deoxyguanosine monophosphate (dGMP), alone or together, canbe used in the method of the invention. One deoxyribonucleosidemonophosphate (i.e., dCMP, dAMP, dTMP or dGMP) and mixtures of two ormore deoxyribonucleoside monophosphates can be used in the method of theinvention.

Preferred dosages of the deoxyribonucleoside monophosphates are betweenabout 100 and about 1,000 mg/kg/day, more preferably between about 200and about 800 mg/kg/day, and most preferably between about 250 and about400 mg/kg/day. If the composition comprises a single deoxyribonucleosidemonophosphate, then the dosages are of the single deoxyribonucleosidemonophosphate. If the composition comprises more than onedeoxyribonucleoside monophosphate, the dosages can be of eachdeoxyribonucleoside monophosphate or of the total deoxyribonucleosidemonophosphates in the composition.

Administration of the deoxyribonucleoside monophosphates can be oncedaily, twice daily, three times daily, four times daily, five timesdaily, up to six times daily, preferably at regular intervals.

Preferred methods of administration are oral, intrathecal, intravenous,and enteral.

Administration of the deoxyribonucleoside monophosphates should begin assoon as the mitochondrial disorder is suspected and continue throughoutthe life of the patient. Test for the diagnosis of such disordersincluding TK2 deficiency are known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted indrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 shows the efficacy of dCMP/dTMP on clinical phenotype in treatedTk2^(−/−) mice. FIG. 1A is a graph of body weight versus age in days,and FIG. 1B of survival of untreated and treated Tk2^(−/−) mice (n=7 foreach group) (Tk2^(−/−) versus Tk2^(−/−200dCMP/dTMP), P<0.005; Tk2^(−/−)versus Tk2^(−/−400dCMP/DTMP), P<0.005; Gehan-Breslow-Wilcoxon test).FIGS. 1C, 1D, and 1E are graphs showing the results of open-field testsfor average distance traveled (FIG. 1C), ambulatory and resting times(FIG. 1D), and horizontal (XY axes) and vertical (Z-axis) movements(FIG. 1E) over 10 minutes in 29-day-old mutant and control wild-typemice treated with 200 mg/kg/day or 400 mg/kg/day of dCMP/dTMP (n=5)(Data expressed as mean±SD. Statistical analysis were performed onTk2^(−/−200dCMP/dTMP) versus Tk2+^(200dCMP/dTMP) andTk2^(−/−400dCMP/dTMP) versus Tk2+^(400dCMP/dTMP)).

FIG. 2 shows the neuromuscular phenotype in 29-day old treated mice.FIG. 2A shows body size, quadriceps muscle size, and myofiber diametersin Tk2^(−/−200dCMP/dTMP (right panels) versus Tk)2^(+200dCMP/dTMP).FIGS. 2B and 2C show body size, quadriceps muscle size, and myofiberdiameters in Tk2^(−/−400dCMP/dTMP) versus Tk2^(+400dCMP/dTMP). FIG. 2Dshows histochemical analysis of muscle cytochrome c oxidasehistochemical activity in Tk2^(−/−200dCMP/dTMP) versusTk2^(+200dCMP/dTMP). FIG. 2E shows a representative western blot ofmitochondrial proteins in the muscles of Tk2^(−/−200dCMP/dTMP) versusTk2^(+200dCMP/dTMP) and percentages of various proteins relative towild-type muscle. FIG. 2F is a table of mitochondrial respiratory chainactivities of Tk2^(−/−200dCMP/dTMP) versus Tk2^(+200dCMP/dTMP) expressedin micromole/min/mg tissue and normalized to mg-protein and relative tocitrate synthase (CS) (mean±SD). No statistically significantdifferences were detected between Tk2^(−/−200dCMP/dTMP) versusTk2^(+200dCMP/dTMP) and Tk2^(−/−400dCMP/dTMP) versus Tk2^(+400dCMP/dTMP)samples.

Abbreviations: CS=citrate synthase; I=NADH-dehydrogenase; II=succinatedehydrogenase; III=cytochrome c reductase; IV=cytochrome c oxidase;V=ATP synthase; I+III=NADH-cytochrome c reductase; II+III=succinatecytochrome c reductase.

FIG. 3 shows the images of results of dCMP/dTMP effects on brain andspinal cord morphology. FIGS. 3A and 3B show hematoxylin and eosin stainshowing numerous vacuoles in 13-day-old untreated Tk2^(−/−)in brain(FIG. 3A) and spinal cord neurons (FIG. 3B). Vacuoles were rare orabsent in Tk2^(−/−200dCMP/dTMP) and not observed in wild-type mice.

FIG. 4 shows the complex I immunohistochemistry and complex IVhistochemistry of the cerebellum. FIGS. 4A-4D show images of complex IV(COX) histochemistry of cerebellum showing deficiency in 13-day-olduntreated Tk2^(−/−) (FIG. 4A) in contrast to normal COX activity in Tk2⁺(FIG. 4B), Tk2^(−/−200dCMP/dTMP) (FIG. 4C), and Tk2^(+200dCMP/dTMP)(FIG. 4D) mice. FIGS. 4E-4H show COX histochemistry (FIGS. 4E and 4F)and immunostaining against COX subunit II (FIGS. 4G and 4H) ofcerebellum of 29-day-oldTk2^(−/−200dCMP/dTMP (upper panels) and age-matched Tk)2^(+200dCMP/dTMP)mice (lower panels). FIGS. 4I and 4J show anti-complex I NDUFB8 subunitimmunostaining of brain staining in 29-day-old Tk2^(−/−200dCMP/dTMP)(FIG. 4I) versus Tk2^(+200dCMP/dTMP) mice (FIG. 4J).

FIG. 5 shows a graphical representation of dCMP/dTMP effects on dNTPpool balance. FIG. 5A shows proportions of dNTPs (in percents) inisolated mitochondria of brain, and FIG. 5B in the liver, of 13 and 29postnatal day mice (P13 and P29) demonstrating that levels of dTTP (redsections) were increased in treated mutant versus untreated mutant miceat P13, but were severely decreased in P29 Tk2^(−/−200dCMP/dTMP) versusTk2^(+200dCMP/dTMP) (*P<0.05; ***P<0.0005; Mann-Whitney U-test).

FIG. 6 shows graphs of mtDNA copy numbers in mice. FIG. 6A showsbaseline mtDNA copy numbers in various tissues, and mtDNA copy numbersin the same tissues in untreated and treated mutants at P13(Tk2^(−/−)versus Tk2^(−/−200dCMP/dTMP); *P<0.05; **P<0.005; ***P<0.0005;Mann-Whitney U-test). FIG. 6B shows mtDNA copy number in various tissuesof treated mutant mice at P29 (expressed as percent of untreatedTk2⁺controls; mean±SD; Tk2^(−/−200dCMP/dTMP) versusTk2^(−/−400dCMP/dTMP); *P<0.05; Mann-Whitney U-test) (n=5 for eachgroup).

FIG. 7 shows the efficacy of dCMP/dTMP on brain hemisphere andcerebellum biochemistry. FIG. 7A shows a graph of CS and complex IVactivity in the cerebral hemispheres of untreated Tk2^(−/−) andTk2^(−/−200dCMP/dTMP) mice relative to untreated wild types(micromole/min/mg tissue normalized to mg-protein; mean±SD). FIG. 7B isa graph of complexes IV and I+III activities when referred to CS(mean±SD) in the cerebral hemispheres of untreated Tk2^(−/−) andTk2^(−/−200dCMP/dTMP) mice relative to untreated wild types(micromole/min/mg tissue normalized to mg-protein; mean±SD). FIG. 7C isa graph of the activities of mitochondrial RC referred to CS (expressedas percent of Tk2+) in the cerebellum of mutant mice at ages 13 and 29days, treated and untreated. FIG. 7D is a western blot of OXPHOS protein(MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail, MitoSciences®)in brain 13-day-old untreated Tk2^(−/−), 13- and 29-day-oldTk2^(−/−200dCMP/dTMP) mice and FIG. 7E in the cerebellum of 13-day-olduntreated Tk2^(−/−), 13- and 29-day-old Tk2^(−/−200dCMP/dTMP) mice, andTk2^(−/−400dCMP/dTMP) (expressed as percentages relative to Tk2⁺). FIGS.7F, 7G, and 7H are graphical quantitation of western blot bands.

Statistical analyses were performed using Tk2^(−/−)versusTk2^(−/−200dCMP/dTMP) with P13 cerebral samples (FIG. 7F);Tk2^(−/−200dCMP/dTMP) versus Tk2^(+200dCMP/dTMP) with P29 cerebralsamples (FIG. 7G); Tk2^(−/−)versus Tk2^(−/−200dCMP/dTMP) with P13cerebellar samples (FIG. 7H); and Tk2^(−/−200dCMP/dTMP) versusTk2^(−/−400dCMP/dTMP) with P29 cerebellar samples (H). *P<0.05;**P<0.005; Mann-Whitney U-test and Unpaired t-test with Welch'scorrection).

Abbreviations: CS=citrate synthase; IV=cytochrome c oxidase (COX);I+III=NADH-cytochrome c reductase; I=NADH-dehydrogenase; II=succinatedehydrogenase; III=cytochrome c reductase; V=ATP synthase; P=postnatalday.

FIG. 8 is graphs showing the metabolism of dCMP/dTMP. FIGS. 8A-8C aregraphs showing the levels of deoxyuridine and deoxythymidine in liver(FIG. 8A), brain (FIG. 8B), and muscle tissues (FIG. 8C) in untreatedmutant mice and treated mutant mice at P13 and P29. Deoxynucleosidelevels are expressed as percentages relative to age-matched untreatedwild-type controls (mean±SD) (Tk2^(−/−200dCMP/dTMP),Tk2^(+200dCMP/dTMP), Tk2−/−400dCMP/dTMP, and Tk2^(+400dCMP/dTMP) versusuntreated Tk2⁺; *P<0.05;**P<0.005; Unpaired t-test with Welch'scorrection; n>3 mice for each group). FIG. 8D is a graph showingthymidine phosphorylase (TP) activity in small intestine of treated anduntreated Tk2 mice at age 29 days relative to 13 days. Data expressed asnmol/h/mg-proteins (mean±SD). FIGS. 8E and 8F are graphs of Tk1 and Tk2activities in brain (FIG. 8E) and muscle tissues (FIG. 8F) of treatedand untreated mice showing increased Tk1 activity in treated mice. Dataexpressed in pmol/min/mg-proteins (mean±SD) (Tk2^(+/+) versusTk2^(+/+200dCMP/dTMP), Tk2^(+/−) versus Tk2^(+/+200dCMP/dTMP), Tk2^(−/−)versus Tk2^(−/−200dCMP/dTMP); *P<0.05; Unpaired t-test with Welch'scorrection; n>3 mice for each group). P=p-value.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is based upon the surprising discovery thatmitochondrial DNA depletion syndromes, including TK2 deficiency, can betreated with a molecular bypass approach. As shown by the resultsherein, the administration of deoxyribonucleoside monophosphates greatlyimproved the condition in both a mouse model of TK2 deficiency and humanpatients with TK2 deficiency.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the methods of the invention and howto use them. Moreover, it will be appreciated that the same thing can besaid in more than one way. Consequently, alternative language andsynonyms may be used for any one or more of the terms discussed herein,nor is any special significance to be placed upon whether or not a termis elaborated or discussed herein. Synonyms for certain terms areprovided. A recital of one or more synonyms does not exclude the use ofthe other synonyms. The use of examples anywhere in the specification,including examples of any terms discussed herein, is illustrative only,and in no way limits the scope and meaning of the invention or anyexemplified term. Likewise, the invention is not limited to itspreferred embodiments.

The term “subject” as used in this application means mammals. Mammalsinclude canines, felines, rodents, bovine, equines, porcines, ovines,and primates. Thus, the invention can be used in veterinary medicine,e.g., to treat companion animals, farm animals, laboratory animals inzoological parks, and animals in the wild. The invention is particularlydesirable for human medical applications

The term “patient” as used in this application means a human subject. Insome embodiments of the present invention, the “patient” is known orsuspected of having mitochondrial disease, mitochondrial DNA depletionsyndrome, or TK2 deficiency.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to cause an improvement in a clinically significantcondition in the subject, or delays or minimizes or mitigates one ormore symptoms associated with the disease or disorder, or results in adesired beneficial change of physiology in the subject.

The terms “treat”, “treatment”, and the like refer to a means to slowdown, relieve, ameliorate or alleviate at least one of the symptoms ofthe disease or disorder, or reverse the disease or disorder after itsonset.

The terms “prevent”, “prevention”, and the like refer to acting prior toovert disease or disorder onset, to prevent the disease or disorder fromdeveloping or minimize the extent of the disease or disorder, or slowits course of development.

The term “in need thereof” would be a subject known or suspected ofhaving or being at risk of having mitochondrial disease, mitochondrialDNA depletion syndrome, or TK2 deficiency.

The term “agent” as used herein means a substance that produces or iscapable of producing an effect and would include, but is not limited to,chemicals, pharmaceuticals, biologics, small organic molecules,antibodies, nucleic acids, peptides, and proteins.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system, i.e., thedegree of precision required for a particular purpose, such as apharmaceutical formulation. For example, “about” can mean within 1 ormore than 1 standard deviations, per the practice in the art.Alternatively, “about” can mean a range of up to 20%, preferably up to10%, more preferably up to 5%, and more preferably still up to 1% of agiven value. Alternatively, particularly with respect to biologicalsystems or processes, the term can mean within an order of magnitude,preferably within 5-fold, and more preferably within 2-fold, of a value.Where particular values are described in the application and claims,unless otherwise stated, the term “about” meaning within an acceptableerror range for the particular value should be assumed.

Deoxypyrimidine Monophosphate Bypass Therapy Mouse Model of TK2Deficiency

Attempts to study the pathogenesis and test therapies for TK2 deficiencyusing cultured fibroblasts from patients had proved unsuccessful becausethe replicating cells failed to manifest mtDNA depletion. Thus, toelucidate the molecular pathogenesis of TK2 deficiency, a homozygous Tk2H126N knock-in mutant (Tk2^(−/−)) mouse that manifests a phenotypestrikingly similar to the human infantile encephalomyopathy wasgenerated and previously reported by the inventors (Akman, et al. 2008)(Example 1).

Based upon the understanding of the pathogenesis of Tk2 deficiency, arationale therapeutic strategy to bypass the enzymatic defect with oraldCMP and dTMP supplementation was designed and implemented (Example 1).Suiprisingly, the results herein demonstrate that the molecular bypasstherapy with orally administered dCMP and dTMP delayed disease onset,ameliorated the abnormal phenotype and extended the lifespan ofTk2^(−/−) mice by 2-3 fold. Additionally, no adverse side effects,including malignancies, were observed (Example 2; FIGS. 1 and 2).

Oral dTMP/dCMP crossed biological barriers including the bloodbrainbarrier (BBB) because treatment increased dTTP in brain and liver in13-day-old Tk2^(−/−) mice and augmented levels of mtDNA, restoring themitochondrial RC activities and protein defects in brain, heart, muscle,liver, and kidney of 13- and 29-day-old mutant mice (Examples 4 and 5;FIGS. 5 and 6). Of particular relevance is the observation thattreatment caused marked improvements of mtDNA levels and biochemicaldefects in muscle, because muscle is the most affected tissue in TK2deficient patients.

Previous studies have found that wild-type Tk2 (mitochondrial) activityis constant in the second week of life while cytosolic Tk1 activitydecreases significantly between postnatal day 8 and 13. Thedown-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2^(−/−) miceand correlates with the onset of mtDNA depletion in brain and heart(Dorado, et al. 2011). The results herein demonstrated that oral dCMPand dTMP delayed the reduction in Tk1 activity (Example 6; FIGS. 8E and8F). Thus, in addition to providing substrates for dNTP synthesis,dCMP/dTMP supplementation in Tk2^(−/−) mice also enhanced thecompensatory Tk1 activity.

The data in mice shown herein proves that deoxypyrimidinesupplementation for Tk2 deficiency is the first effective and safe invivo treatment option for patients affected by Tk2 mutations. SeeGarone, et al. 2014, herein incorporated by reference in its entirety.Based upon this data, the administration of deoxyribonucleosidemonophosphates was hypothesized to improve the conditions of humanpatients with TK2 deficiency, even though mouse models have revealedimportant differences in dNTP homeostasis between mice and humans.

Furthermore, this approach is applicable to individuals with othermitochondrial disorders due to nucleotide pools unbalance.

Patients Benefitting from the Administration of DeoxyribonucleosideMonophosphates

The present invention includes the administration of at least onedeoxyribonucleoside monophosphate to a patient in need thereof. In oneembodiment, the present invention includes the administration of atleast one deoxypyrimidine monophosphate. In a further embodiment, thedexypyrimidine monophosphate is chosen from dCMP, dTMP and mixturesthereof. In yet another embodiment, the present invention includes theadministration of at least one deoxypurine monophosphate. In a furtherembodiment, the deoxypurine monophosphate is chosen from dAMP, dGMP, andmixtures thereof.

As shown in Examples 7-10, the administration of dTMP and dCMP greatlyimproved the symptoms of TK2 deficiency in patients, especially withregard to muscular-related symptoms. Thus, patients who would benefitfrom the deoxypyrimidine monophosphate bypass therapy would be thosediagnosed with TK2 deficiency. In these patients, at least onedeoxypyrimidine monophosphate, dCMP or dTMP, or mixtures thereof wouldbe administered.

A parallel defect of deoxyguanosine kinase (dGK), due to autosomalrecessive mutations in DGUOK with deficiencies in dGMP and dAMP, causesmtDNA depletion typically manifesting as early childhood-onsethepatocerebral disease (Mandel et al., 2001). These patients wouldbenefit from the administration of at least one deoxypurinemonophosphates, dGMP or dAMP, or mixtures thereof.

Other forms of MDS as well as other disorders related to unbalancednucleotide pools can be treated by the administration of specificdeoxyribonucleoside monophosphate, i.e., dAMP, dGMP, dCMP, or dTMP, ormixtures thereof. These disorders would include but are not limited todeficiencies related to RRM2B (encoding p53R2, the p53-inducible smallsubunit of ribonucleotide reductase, RNR) and mutations in TYMP(encoding thymidine phosphorylase, TP) which cause mitochondrialneurogastrointestinal encephalomyopathy (MNGIE).

Additionally, as the mechanisms of other forms of MDS and otherdisorders become elucidated, the proper deoxyribonucleosidemonophosphate(s) for treatment can be determined by the skilledpractitioner.

Patients that exhibit the phenotype discussed above for TK2 deficiencyincluding the most typical presentation of progressive muscle diseasecharacterized by generalized hypotonia, proximal muscle weakness, lossof previously acquired motor skills, poor feeding, and respiratorydifficulties, can be tested to definitively diagnose the disease.

If the clinical presentation is highly suspicious for mtDNA depletionsyndrome, molecular genetic testing using a panel of genes known tocause mtDNA depletion syndrome should be performed (Chanprasert, et al.2012). The TK2 gene is the only gene in which mutations are known tocause TK2-related mitochondrial DNA depletion syndrome. This testing caninclude a sequence analysis of the entire coding and exon/intronjunction regions of TK2 for sequence variants and deletion/duplication.If compound heterozygous or homozygous deleterious mutations areidentified in the sequence analysis, the diagnosis of TK2 deficiency isconfirmed, and thus, the subject would benefit from the deoxypyrimidinemonophosphate therapy. If sequence analysis does not identify twocompound heterozygous or homozygous deleterious mutations,deletion/duplication analysis should be considered to determine and/orconfirm a TK2 deficiency diagnosis.

Further tests to determine and/or confirm a TK2 deficiency diagnosis mayinclude testing serum creatine kinase (CK) concentration,electromyography, histopathology on skeletal muscle, mitochondrial DNA(mtDNA) content (copy number), and electron transport chain (ETC)activity in skeletal muscle. If one or more of the following is found inthese tests, the TK2 deficiency is determined and/or confirmed. ElevatedCK concentration as compared to healty controls can indicate TK2deficiency. A skeletal muscle biopsy can be performed, and then a mtDNAcontent analysis in skeletal muscle performed. If the skeletal musclebiopsy shows prominent variance in fiber size, variable sarcoplasmicvacuoles, variable increased connective tissue, and ragged red fibers aswell as increased succinate dehydrogenase (SDH) activity and low toabsent cytochrome c oxidase (COX) activity, and mtDNA copy number isseverely reduced (typically less than 20% of age- and tissue-matchedhealthy controls), a diagnosis of TK2 deficiency can be determinedand/or confirmed (Chanprasert, et al. 2012).

Additionally, TK2 deficiency is inherited in an autosomal recessivemanner. Thus, a sibling of an affected patient can be tested as early aspossible after birth to diagnose the disease.

In all of these examples, deoxypyrimidine monophosphate bypass therapyshould be started as soon as possible after a diagnosis of TK2deficiency.

Pharmaceutical Compositions, Methods of Administration, and Dosing

The present invention encompasses the administration ofdeoxyribonucleoside monophosphates. Most preferred methods ofadministration are oral, intrathecal and parental including intravenous.The deoxyribonucleoside monophosphates must be in the appropriate formfor administration of choice.

The preferred form of the administration of the deoxyribonucleosidemonophosphates is in the form of disodium salts of thedeoxyribonucleoside monophosphates. These disodium salts (dTMP Na2, dCMPNa₂ salts) are easily dissolved in liquid (such as water, formula ormilk) whereas the free acid form does not readily dissolve in liquid.Once administered, the deoxyribonucleoside monophosphates salts convertto the free acid form. By way of example, for some of the oraladministration to human patients, 2000 mg each of dTMP Na₂ and dCMP Na₂were dissolved in 25 ml of water.

Such compositions for administration may comprise a therapeuticallyeffective amount of the deoxyribonucleoside monophosphates and apharmaceutically acceptable carrier. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that arephysiologically tolerable and do not typically produce an allergic orsimilar untoward reaction, such as gastric upset, dizziness and thelike, when administered to a human, and approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

“Carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as saline solutions in water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.A saline solution is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol, and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

Oral administration is a preferred method of administration. Thedeoxyribonucleoside monophosphates can be added to any form of liquid apatient would consume including but not limited to, milk, both cow's andhuman breast, infant formula, and water.

Additionally, pharmaceutical compositions adapted for oraladministration may be capsules, tablets, powders, granules, solutions,syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions.Tablets or hard gelatin capsules may comprise lactose, starch orderivatives thereof, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, stearic acid or salts thereof. Soft gelatincapsules may comprise vegetable oils, waxes, fats, semi-solid, or liquidpolyols. Solutions and syrups may comprise water, polyols, and sugars.An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract. Thus, the sustainedrelease may be achieved over many hours and if necessary, the activeagent can be protected from degradation within the stomach.Pharmaceutical compositions for oral administration may be formulated tofacilitate release of an active agent at a particular gastrointestinallocation due to specific pH or enzymatic conditions.

In order to overcome issue of the deoxynucleoside monophosphatescrossing the blood/brain barrier, intrathecal administration is afurther preferred form of administration of deoxyribonucleosidemonophosphates (Galbiati, et al. 2006; Gotz, et al. 2008). Intrathecaladministration involves injection of the drug into the spinal canal,more specifically the subarachnoid space such that it reaches thecerebrospinal fluid. This method is commonly used for spinal anesthesia,chemotherapy, and pain medication. Intrathecal administration can beperformed by lumbar puncture (bolus injection) or by a port-cathetersystem (bolus or infusion). The catheter is most commonly insertedbetween the laminae of the lumbar vertebrae and the tip is threaded upthe thecal space to the desired level (generally L3-L4). Intrathecalformulations most commonly use water, and saline as excipients but EDTAand lipids have been used as well.

A further preferred form of administration is parenteral includingintravenous administration. Pharmaceutical compositions adapted forparenteral administration, including intravenous administration, includeaqueous and non-aqueous sterile injectable solutions or suspensions,which may contain anti-oxidants, buffers, bacteriostats, and solutesthat render the compositions substantially isotonic with the blood ofthe subject. Other components which may be present in such compositionsinclude water, alcohols, polyols, glycerine, and vegetable oils.Compositions adapted for parental administration may be presented inunit-dose or multi-dose containers, such as sealed ampules and vials,and may be stored in a freeze-dried (lyophilized) condition requiringonly the addition of a sterile carrier, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules, and tablets. Suitable vehicles that can beused to provide parenteral dosage forms of the invention are well knownto those skilled in the art. Examples include: Water for Injection USP;aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, and LactatedRinger's Injection; water-miscible vehicles such as ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Additionally, since some patients may be receiving enteral nutrition bythe time the deoxyribonucleoside monophosphate treatment begins, thedNMPs can be administered through a gastronomy feeding tube or otherenteral nutrition means.

Further methods of administration include mucosal, such as nasal,sublingual, vaginal, buccal, or rectal; or transdermal administration toa subject.

Pharmaceutical compositions adapted for nasal and pulmonaryadministration may comprise solid carriers such as powders, which can beadministered by rapid inhalation through the nose. Compositions fornasal administration may comprise liquid carriers, such as sprays ordrops. Alternatively, inhalation directly through into the lungs may beaccomplished by inhalation deeply or installation through a mouthpiece.These compositions may comprise aqueous or oil solutions of the activeingredient. Compositions for inhalation may be supplied in speciallyadapted devices including, but not limited to, pressurized aerosols,nebulizers or insufflators, which can be constructed so as to providepredetermined dosages of the active ingredient.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient over a prolonged period of time.

Selection of a therapeutically effective dose will be determined by theskilled artisan considering several factors, which will be known to oneof ordinary skill in the art. Such factors include the particular formof the deoxyribonucleoside monophosphate, and its pharmacokineticparameters such as bioavailability, metabolism, and half-life, whichwill have been established during the usual development procedurestypically employed in obtaining regulatory approval for a pharmaceuticalcompound. Further factors in considering the dose include the conditionor disease to be treated or the benefit to be achieved in a normalindividual, the body mass of the patient, the route of administration,whether the administration is acute or chronic, concomitant medications,and other factors well known to affect the efficacy of administeredpharmaceutical agents. Thus, the precise dose should be decidedaccording to the judgment of the person of skill in the art, and eachpatient's circumstances, and according to standard clinical techniques.

A preferred dose ranges from about 100 mg/kg/day to about 1,000mg/kg/day. A further preferred dose ranges from about 200 mg/kg/day toabout 800 mg/kg/day. A further preferred dose ranges from about 250mg/kg/day to about 400 mg/kg/day. These dosage amounts are of individualdeoxyribonucleoside monophosphates or of a composition with a mixture ofmore than one deoxyribonucleoside monophosphates, e.g., dTMP and dCMP.For example, a dose can comprise 400 mg/kg/day of dTMP alone. In afurther example, a dose can comprise a mixture of 200 mg/kg/day of dTMPand 200 mg/kg/day of dCMP. In a further example, a dose can comprise 400mg/kg/day of a mixture of dTMP and dCMP.

Administration of the deoxyribonucleoside monophosphates can be once aday, twice a day, three times a day, four times a day, five times a day,up to six times a day, preferably at regular intervals. For example,when the deoxyribonucleoside monophosphates are administered four timesdaily, doses would be at 8:00 AM, 12:00 PM, 4:00 PM, and 8:00 PM.

As shown in the Examples, doses can be adjusted to optimize the effectsin the subject. For example, the deoxyribonucleoside monophosphates canbe administered at 100 mg/kg/day to start, and then increased over timeto 200 mg/kg/day, to 400 mg/kg/day, to 800 mg/kg/day, up to 1000mg/kg/day, depending upon the subject's response.

A subject can be monitored for improvement of their condition prior toincreasing the dosage. Also as shown in the Examples, a subject'sresponse to the therapeutic administration of the deoxyribonucleosidemonophosphates can be monitored by observing a subject's muscle strengthand control, and mobility as well as changes in height and weight. Ifone or more of these parameters increase after the administration, thetreatment can be continued. If one or more of these parameters stays thesame or decreases, the dosage of the deoxyribonucleoside monophosphatescan be increased.

The deoxyribonucleoside monophosphates can also be co-administered withother agents. Such agents would include therapeutic agents for treatingthe symptoms of the particular form of MDS. In particular, for TK2deficiency, the dTMP and dCMP can be co-administered with an inhibitorof thymidine phosphorylase (e.g. tipiracil) or an inhibitor of cytidinedeaminase (e.g. tetrahydrouridine [THU]) (see Example 6). Suchinhibitors are known and used in the treatment of some cancers.

EXAMPLES

The present invention may be better understood by reference to thefollowing non-limiting examples, which are presented in order to morefully illustrate the preferred embodiments of the invention. They shouldin no way be construed to limit the broad scope of the invention.

Example 1 Materials and Methods for Examples 2-6 Mouse Model of TK2Deficiency

A homozygous Tk2 H126N knock-in mutant (Tk2^(−/−)) mouse that manifestsa phenotype strikingly similar to the human infantile encephalomyopathyhas been previously reported (Akman, et al. 2008). Between postnatal day10 and 13, Tk2^(−/−) mice rapidly develop fatal encephalomyopathycharacterized by decreased ambulation, unstable gait, coarse tremor,growth retardation, and rapid progression to early death at age 14 to 16days. Molecular and biochemical analyses of the mouse model demonstratedthat the pathogenesis of the disease is due to loss of enzyme activityand ensuing dNTP pool imbalances with decreased dTTP levels in brain andboth dTTP and dCTP levels in liver, which, in turn, produces mtDNAdepletion and defects of respiratory chain enzymes containingmtDNA-encoded subunits, most prominently in the brain and spinal cord(Dorado, et al, 2011).

All experiments were performed according to a protocol approved by theInstitutional Animal Care and Use Committee of the Columbia UniversityMedical Center, and were consistent with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals. Mice werehoused and bred according to international standard conditions, with a12-hour light, 12-hour dark cycle, and sacrificed at 4, 13, and 29 daysof age.

Organs (brain, spinal cord, liver, heart, kidney, quadriceps muscle,lung, and gastrointestinal tract) were removed and either frozen in theliquid phase of isopentane, pre-cooled near its freezing point (−160°C.) with dry ice or fixed in 10% neutral buffered formalin and embeddedin paraffin using standard procedures. Paraffin embedded tissue werethen stained with hematoxylin and eosin (H&E) for morphological study orprocessed for immunostaining studies with GFAP, COX I, or complex Isubunit as detailed below. All the experiments were performed in atleast 3 mice per group. Both heterozygous and homozygous wild type micewere considered as control group (Tk2⁺) since no clinical andbiochemical difference were previously described (Akman et al, 2008;Dorado et al, 2011).

Treatment Administration and Experimental Plan

Deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate(dTMP) (Hongene Biotech, Inc.) were administered in 50 μl of Esbilacmilk formula for small pets (Pet-Ag) by daily oral gavage to Tk2 H126Nknock-in mice (Tk2^(−/−)) and aged-matched control wild-type (Tk2⁺)using 2 doses, 200 mg/kg/day and 400 mg/kg/day, from post-natal day 4 to29 days. At age 29 days, mice were separated from the mother and thetreatment was continued by administration of dCMP and dTMP in drinkingwater using equimolar doses, respectively of 1.6 mM and 3.2 mM. Anegative control group of untreated Tk2 mutant and control wild-typemice were weighted and observed closely for comparison.

Phenotype Assessment

To define the degree of safety and efficacy of dTMP/dCMP, survival time,age-at-onset of disease, type and severity of symptoms, occurrence ofside effects, and proportion of treatment termination due to adverseevents, in treated and untreated Tk2 mice were compared. Generalbehavior, survival time, and body weights of the mice were assesseddaily beginning at postnatal day 4. Videotaping and open-field test withan Opto-Varimetrix-3 sensor system (Columbus Instruments) were performedat 13 and 29 days by counting horizontal and vertical movements,recording ambulatory and resting time, and measuring the total distancetraveled in 10 minutes.

Brain Histology

Brain and spinal cord samples from 13- to 29-day-old mice were fixedwith 10% neutral-buffered formalin and embedded in paraffin usingstandard procedures. Cerebellum, brainstem, hippocampus, cerebralcortex, and cervical, thoracic and lumbar tracts of the spinal cord wereanalyzed.

Sections (5 μm thick) were stained with H&E and luxol fast blue toanalyze the overall structure of the tissue. Immunostaining withantibodies against GFAP, complex I (NDUFB6), or COX subunit 2 was alsoperformed. Briefly, paraffin-embedded brain and spinal cord slides weredeparaffinized, rehydrated, and rinsed in phosphate-buffered salinesolution (PBS). To block endogenous peroxidase activity, sections wereincubated with 3% hydrogen peroxide in methanol. Slides were then placedin 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave ovenfor 15 minutes, for antigen retrieval. Slides were incubated with mouseanti-GFAP antibody (1:100) (Novocastra. NCL-GFAP-GA5) or mousemonoclonal antibody anti-complex I 17 kDa (NDUFB6) subunit (1:100)(A21359; Molecular Probes) or mouse monoclonal antibody anti-COX subunit2 (1:100) (clone COX 229, A6404; Molecular Probes) overnight at 4° C.Sections were subsequently rinsed in PBS and incubated with anti-mouseM.O.M. Peroxidase kit, 1:200 dilution for 60 minutes at roomtemperature. Immunoreactivity was detected by avidin-biotin complex(ABC) with DAB substrate (Vector Laboratories, Burlingame, Calif., USA).Slides were examined by light microscopy using an Olympus BX51microscope, and images were captured with a QImaging Retiga EXi digitalcamera, using QCapture software version 2.68.6.

dNTP Pool by Polymerase Extension Assay

Tissues were homogenized on ice in 10 volumes (w/v) of cold MTSE buffer(210 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl pH 7.5, 0.2 mM EGTA,0.5% BSA) and centrifuged at 1,000 g for 5 minutes at 4° C., followed bythree centrifugations at 13,000 g for 2 minutes at 4° C. Supernatant wasprecipitated with 60% methanol for the mitochondrial fraction and 100%methanol for the cytosolic fraction, kept 2 hours at −80° C., boiled 3minutes, stored at −80° C. (from 1 hour to overnight), and centrifugedat 20,800 g for 10 minutes at 4° C. Supernatants were evaporated untildry, and pellet was resuspended in 65 μl of water and stored at −80° C.until analyzed.

To minimize ribonucleotide interference, total dNTP pools weredetermined as previously reported (Ferraro, et al. 2010; Marti, et al.2012). Briefly, 20 μl volume reactions was generated by mixing 5 μl ofsample or standard with 15 μl of reaction buffer (0.025 U/mlThermoSequenase DNA polymerase (GE Healthcare, Piscataway, N.J., USA) orTaq polymerase (Life Technologies, NY, USA), 0.75 μM ³H-dTTP or ³H-dATP(Moravek Biochemicals), 0.25 μM specific oligonucleotide, 40 mM TrisHCl,pH 7.5, 10 mM MgCl₂, 5 mM DTT). After 60 minutes at 48° C., 18 ml ofreaction were spotted on Whatman DE81 filters, air dried, and washedthree times for 10 minutes with 5% Na₂HPO₄, once in distilled water andonce in absolute ethanol. The retained radioactivity was determined byscintillation counting.

Nucleosides Measurement by HPLC

Deoxythymidine (dT), deoxyuridine (dU), uracil (U), and thymine (T)levels were assessed by a gradient-elution HPLC method as describedpreviously (Lopez, et al. 2009; Marti, et al. 2012), with minormodifications. Briefly, deproteinized samples were injected into anAlliance HPLC system (Waters Corporation) with an Alltima C18NUCreversed-phase column (Alltech) at a constant flow rate of 1.5 ml/min(except where indicated) using three buffers: eluent A (20 mM potassiumphosphate, pH 5.6), eluent B (water), and eluent C (methanol). Sampleswere eluted over 60 minutes with a gradient as follows: 0-5 min, 100%eluent A; 5-25 min, 100-71% eluent A, 29% eluent B; 25-26 min, 0-100%eluent C; 26-30 min, 100% eluent C; 30-31 min, 0-100% eluent B; 31-35min, 100% eluent B (1.5-2 ml/min); 35-45 min, 100% eluent B (2 ml/min);45-46 min, 100% eluent B (2-1.5 ml/min); 46-47 min, 0-100% eluent C;47-50 min, 100% eluent C; 50-51 min, 0-100% eluent A; and 51-60 min,100% eluent A.

Absorbance of the elutes was monitored at 267 nm, and dThd and dUrdpeaks were quantified by comparing their peak areas with a calibrationcurve obtained with aqueous standards. For definitive identification ofdT, dU, U, and, T peaks for each sample, a second aliquot treated withexcess of purified E. coli TP (Sigma) was used to specifically eliminatedT and dU. The detection limit of this method is 0.05 mmol/l for allnucleosides.

RT-qPCR: Mitochondrial DNA Quantification

Real-time PCR was performed with the primers and probes for murine COX Igene (mtDNA) and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH,nDNA) (Applied Biosystems, Invitrogen, Foster City, Calif., USA) asdescribed using standard curve quantification, in an ABI PRISM 7,000Sequence Detection System (Applied Biosystems) (Dorado, et al. 2011).MtDNA values were normalized to nDNA values and expressed as percentrelative to wild type (100%).

Mitochondrial Respiratory Chain Protein Levels

Thirty micrograms of whole brain cerebrum or cerebellum extracts waselectrophoresed in an SDS-12% PAGE gel, transferred to Immun-BlotTM PVDFmembranes (Bio-Rad, Hercules, Calif., USA) and probed with MitoProfile®Total OXPHOS Rodent WB Antibody Cocktail of antibodies (MitoSciences,Eugene, Oreg., USA). Proteinantibody interaction was detected withperoxidase-conjugated mouse anti-mouse IgG antibody (Sigma-Aldrich, StLouis, Mo., USA), using Amersham™ ECL Plus western blotting detectionsystem (GE Healthcare Life Sciences, UK). Quantification of proteins wascarried out using NIH ImageJ 1.37V software. Average gray value wascalculated within selected areas as the sum of the gray values of allthe pixels in the selection divided by the number of pixels.

Mitochondrial Respiratory Chain Enzyme Activities by SpectrophotometerAnalysis

Mitochondrial RC enzymes analysis was performed in cerebrum andcerebellum tissues as previously described (DiMauro, et al. 1987).

Nucleosides and Nucleotides Metabolic Enzymes

Thymidine phosphorylase and thymidine kinase 1 and 2 activities weremeasured as previously described (Marti, et al. 2003; Lopez, et al.2009; Dorado, et al. 2011).

Statistical Methods

Data are expressed as the mean±SD of at least three experiments pergroup. Gehan-Breslow-Wilcoxon test was used to compare the survivalproportion of each group of mice. Unpaired t-test with Welch'scorrection and Mann-Whitney U-test were used to compare 13-day-old oldTk2⁺ versus untreated Tk2^(−/−), 13-day-old untreated Tk2^(−/−) versusTk2^(−/ −200dCMP/dTMP), 29-day-old wild-type versusTk2^(−/−200dCMP/dTMP) and Tk2^(−/−400dCMP/dTMP), for molecular andbiochemical studies. Response to treatment was evaluated comparingTk2^(−/−) versus Tk2^(−/−200dCMP/dTMP) at 13 days andTk2^(−/−200dCMP/dTMP) versus Tk2^(−/ −400dCMP/dTMP). A P-value of <0.05was considered to be statistically significant.

Example 2 dCMP/dTMP Delays Disease Onset, Prevents NeuromuscularManifestations, and Prolongs Lifespan of Tk2-Deficient Mice

Oral treatment with dCMP+dTMP 200 mg/kg/day each in milk(Tk2^(−/−200dCMP/dTMP)) beginning at postnatal day 4 delayed diseaseonset to 20-25 days when the mutant mice developed a mild tremor andstopped gaining weight (data not shown). In the fourth week, theymanifested weakness and reduced movements. In contrast, Tk2^(−/−) micetreated from day 4 with dCMP+dTMP 400 mg/kg/day each in milk(Tk2^(−/−400dCMP/dTMP)) appeared normal until day 21, when weight gaindecelerated and mild head tremor developed (FIG. 1A).

Untreated Tk2^(−/−) mice had a mean lifespan of 13.2±2.5 days (mean±SD),whereas Tk2^(−/−200dCMP/dTMP) survived to 34.6±3.2 days (P=0.0028; n=7;Gehan-Breslow-Wilcoxon test) while Tk2^(−/−400dCMP/dTMP) lived to44.3±9.1 days (P=0.0071; n=7; Gehan-Breslow-Wilcoxon test) (FIG. 1B).The cause of death was not evident in postmortem histological studies ofmajor organs in 29-day-old Tk2^(−/−200dCMP/dTMP) mice. No adverse sideeffects, including malignancies, were observed in the treatedhomozygous, and heterozygous wild types (Tk2⁺) and mutants except milddeceleration of weight gain in Tk2^(+400dCMP/dTMP) (data not shown).

Open-field assessment of motor function in 29-day-oldTk2^(−/−200dCMP/dTMP), Tk2^(−/−400dCMP/dTMP), and wild-type Tk2 miceshowed no differences in the distance traveled, horizontal and verticalmovements, or resting time (FIGS. 1C-E).

Relative to 29-day-old Tk2⁺ mice, age-matched Tk2^(−/−200dCMP/dTMP) andTk2^(−/−400dCMP/dTMP) animals showed decreases in gross muscle mass andmuscle fiber diameter that were independent of the treatment dose butparalleled to body weight (FIG. 2). Histology showed no signs ofmyopathy or mitochondrial abnormalities (FIGS. 2A-D). Biochemicalstudies demonstrated normal mitochondrial RC activities and proteinlevels (FIGS. 2D-F).

Example 3 Histological and Histochemical CNS Studies Confirmed dCMP/dTMPEfficacy

Efficacy of treatment in central nervous system (CNS) was demonstratedin histological studies that showed dramatic reductions in the numbersof vacuoles in neurons of the spinal cord and cerebellar and brain stemnuclei of 13-day-old Tk2^(−/−200dCMP/dTMP) mice relative to untreated13-day-old Tk2^(−/−) mice (FIGS. 3A and 3B). Furthermore, cytochrome coxidase (COX, complex IV) histochemistry of cerebellum revealed reducedoverall COX activity in 13-day-old untreated Tk2^(−/−) mice (FIG. 4A)with normal activities in 13- and 29-day-old Tk2^(−/−200dCMP/dTMP)(FIGS. 4C and 4E) relative to Tk2⁺ animals (FIGS. 4B, 4D, and 4F). Nocell-specific immunohistochemical differences in COX protein weredetected (FIGS. 4G and 4H) while severe reduction in complex I wasidentified by immunostaining of cerebellum of 29-day-oldTk2^(−/−200dCMP/dTMP) (FIGS. 4I and 4J).

Example 4 Treatment Crosses Biological Barriers

To confirm that the treatment crosses biological barriers, dNTP levelsin isolated mitochondria were assessed by polymerase extension assaydescribed in Example 1. In 13-day-old untreated Tk2^(−/−) mice relativeto Tk2+ littermates, isolated brain mitochondria showed decreased levelsof dTTP (0.67±0.1 pmol/mg-protein versus 2.52±1.0), while isolated livermitochondria revealed reduced dCTP levels (1.07±0.8 versus 2.9±1.0)(Table 1). The treatment crossed the blood-brain barrier increasing thelevel of dTTP in isolated brain mitochondria of 13-day-oldTk2^(+200dCMP/dTMP) (3.55±1) and Tk2^(−/−200dCMP/dTMP) (1.5±0.7) and asa consequence, restored the proportion of dTTP relative to total dNTP intreated mutants. In contrast, levels of dCTP in isolated mitochondriawere stable in brain of 13-day-old Tk2^(+200dCMP/dTMP) (3.07±2),decreased in brain of Tk2^(−/−200dCMP/dTMP) (1.13±0.5), and decreased inliver of 13-day-old Tk2^(+200dCMP/dTMP) (1 .13±0.4) andTk2^(−/−200dCMP/dTMP) (0.56±0.5) (Table 1).

In 29-day-old Tk2^(−/−200dCMP/dTMP) relative to Tk2⁺ mice, absolutelevels of dTTP and dCTP were markedly reduced in isolated mitochondriafrom brain (dTTP 0.11±0.05 and dCTP 0.6±0.2) and from liver (dTTP0.15±0.04 and dCTP 0.04±0.03) (Table 1); when these data were expressedas percentage of total dNTPs, there were striking decreases in dTTP/dNTPin brain (P=0.0322; n=7; Mann-Whitney U-test) and dCTP/dNTP in liver(P=0.0338; n=3; Mann-Whitney U-test) (FIGS. 5A and B).

TABLE 1 dNTP pools level in tissues Untreated Untreated Untreated BrainTk2⁺ Tk2^(−/−) Tk2^(+200dCMP/dTMP) Tk2^(−/−200dCMP/dTMP) Tk2⁺Tk2^(+200dCMP/dTMP) Tk2^(−/−200dCMP/dTMP) Mitochondria (P 13; n = 5) (P13; n = 4) (P 13; n = 5) (P 13; n = 5) (P 29; n = 4) (P 29; n = 5) (P29; n = 8) dATP 0.91 ± 0.2 1.51 ± 1    0.85 ± 0.5 1.03 ± 0.8 0.44 ± 0.2 0.86 ± 0.4 0.5 ± 0.3 dTTP 2.52 ± 1  0.67 ± 0.1* 3.55 ± 1 1.52 ± 0.71.87 ± 0.9 3.5 ± 1  0.11 ± 0.05** dGTP 1.06 ± 0.4 1.17 ± 0.7   1.9 ± 10.68 ± 0.3  1.3 ± 0.5 1.9 ± 1 0.87 ± 0.6  dCTP 1.99 ± 0.9 3.9 ± 3  3.07± 2 1.13 ± 0.5 0.32 ± 0.2  1.2 ± 0.6 0.6 ± 0.2 Untreated UntreatedUntreated Liver Tk2⁺ Tk2^(−/−) Tk2^(+200dCMP/dTMP) Tk2^(−/−200dCMP/dTMP)Tk2⁺ Tk2^(+200dCMP/dTMP) Tk2^(−/−200dCMP/dTMP) Mitochondria (P 13; n =3) (P 13; n = 3) (P 13; n = 3) (P 13; n = 3) (P 29; n = 3) (P 29; n = 5)(P 29; n = 4) dATP 1.47 ± 1  1.34 ± 0.8 1.42 ± 0.8 2.6 ± 1  0.32 ± 0.2 0.5 ± 0.3 0.31 ± 0.3  dTTP  0.61 ± 0.08 0.54 ± 0.2 0.67 ± 0.3 0.36 ±0.3 0.46 ± 0.3  1.2 ± 0.8 0.15 ± 0.04* dGTP 2.2 ± 2 2.64 ± 1  0.93 ± 0.91.2 ± 1   0.58 ± 0.25 0.75 ± 0.4 0.8 ± 0.6  dCTP 2.9 ± 1 1.07 ± 0.8 1.13± 0.4 0.56 ± 0.5 0.36 ± 0.3 0.67 ± 0.2 0.04 ± 0.03* Data expressed inpmol normalized to mg-protein (mean ± SD). Statistical analyses wereperformed with untreated Tk2^(−/−) vs untreated Tk2⁺ (P 13) andTk2^(−/−200dCMP/dTMP) vs Tk2^(+200dCMP/dTMP) (P 13 and P 29). *= p <0.05; **= p < 0.005. P = postnatal day

Example 5 dCMP/dTMP Treatment Ameliorates Biochemical and MolecularGenetic Abnormalities

Treatment with dCMP and dTMP enhanced mtDNA levels in the mutant mice.At pre-treatment baseline, 4-day-old Tk2^(−/−) mice did not manifestclinical abnormalities, but showed reductions of mtDNA copy numbers inbrain cerebrum (38±13% mtDNA relative to wild-type brain, P=0.0002; n=5;Mann-Whitney U-test), cerebellum (54±1%, P=0.0228; n=4; Mann-WhitneyU-test), muscle (28±12%), and kidney (62±11%) with normal mtDNA levelsin heart and liver (FIG. 6A). At age 13 days, untreated Tk2^(−/−)animals showed marked mtDNA depletion in brain cerebrum (21±3%,P<0.0025; n=5; Mann-Whitney U-test), muscle (47±1%, P=0.0303; n=7;Mann-Whitney U-test), liver (32±1%, P=0.0140; n=5; Mann-Whitney U-test),and kidney (35±9%, P=0.008; n=6; Mann-Whitney U-test), but stable mtDNAdepletion in the cerebellum (FIG. 6A). In contrast, with treatment,13-day-old Tk2^(−/−2dCMP/dTMP) mice manifested moderate mtDNA depletiononly in brain cerebrum (66±34%) and normal mtDNA levels in cerebellum,muscle, heart, liver, and kidney (FIG. 6A).

At age 29 days, relative to Tk2+ mice, Tk2^(−/−200dCMP/dTMP) mice showedmtDNA depletion that was severe in cerebellum (23±8%) and brain cerebrum(11±1%) and moderate in muscle (48±23%), liver (70±13%), and kidney(55±6%) (FIG. 6B). Compared with Tk2^(−/−200dCMP/dTMP) mice,Tk2^(−/−400dCMP/dTMP) animals had less severe mtDNA depletion in braincerebrum (22±8%, P=0.0159; n=6; Mann-Whitney U-test), but similar mtDNAdepletion in muscle (40±8%), liver (71±36%), and kidney (43±11%) andcerebellum (26±12%) (FIG. 6B).

To assess the impact of treatment on mitochondrial RC enzymes, theiractivities and steady-state protein levels in brain cerebrum andcerebellum were measured. In 13-day-old untreated Tk2^(−/−) mice,relative to untreated wild-type, brain cerebrum showed reduced COXactivity (57±19%, P=0.0159; n=5; Mann-Whitney U-test) and significantlyincreased citrate synthase (CS) activity (148±17%; P=0.0317; n=5;Mann-Whitney U-test) (Table 2; FIG. 7A) and, when normalized to CS,revealed decreased activities of complexes I+III (NADH-cytochrome creductase) (76±0.06%, P=0.0159; n=5; Mann-Whitney U-test) and II+III(succinate-cytochrome c reductase) (72±9%) in addition to IV (41±14%,P=0.0079; n=5; Mann-Whitney U-test) (Table 2; FIG. 7B).

The RC defects were more severe in cerebellum with significantreductions in all of the complexes when normalized either to CS (FIG.7C) or to mg-proteins with predominant defect in complex I (29±15%;P=0.0087; n=5; Unpaired t-test with Welch's correction) and increased CSactivity (129±34%) (Table 3). In contrast, 13-day-oldTk2^(−/−200dCMP/dTMP) had normal RC enzyme activities in brain cerebrum(Table 2; FIGS. 7A and 7B) and only a mild defect in complex I (56±21%)in cerebellum compared with age-matched treated control mice (Table 3;FIG. 7C). In 29-day-old Tk2^(−/−200dCMP/dTMP), activities of RC enzymeswere normal in brain cerebrum (Table 2). In contrast, cerebellum ofTk2^(−/−200dCMP/dTMP) manifested a mild defect in complex IV (62±20%)and severe defect in complex I+III (35±24%, P=0.0296; n=5; Mann-WhitneyU-test), while RC activities were completely rescued in theTk2^(−/−400dCMP/dTMP) (Table 3; FIG. 7C).

Western blot analysis of mitochondrial RC complex subunits in 13-day-olduntreated Tk2^(−/−) animals compared with untreated control micerevealed reductions in steady-state levels of complex I (55±39% braincerebrum; 38±13% cerebellum, P=0.025; n=6; Mann-Whitney U-test) andcomplex IV (74±32% brain cerebrum; 44±16% cerebellum, P=0.0017; n=6;Mann-Whitney U-test), while RC protein levels were normal in 13-day-oldTk2^(−/−200dCMP/dTMP) mice (FIGS. 7D-7F and 7H). In 29-day-oldTk2^(−/−200dCMP/dTMP) compared with Tk2^(+200dCMP/dTMP), a reducedlevels of subunits of complexes I (45%±2, P=0.0196; n=4; Mann-WhitneyU-test) and III (69%±2) were observed in brain cerebrum (FIG. 7G) andcomplexes I (34%±4) and IV (49%±1, P=0.0267; n=4; Unpaired t-test withWelch correction) in cerebellum (FIG. 7H). In 29-day-oldTk2^(−/−400dCMP/dTMP), a complete rescue of the RC defect was observedwhen compared to Tk2^(+400dCMP/dTMP).

TABLE 2 Mitochondrial respiratory chain enzyme activities in brainhemisphere Untreated Untreated Tk2⁺ Tk2^(−/−) Tk2^(+200dCMP/dTMP)Tk2^(−/−200dCMP/dTMP) Tk2^(+200dCMP/dTMP) Tk2^(−/−200dCMP/dTMP) (P 13; n= 5) (P 13; n = 5) (P 13; n = 4) (P 13; n = 5) (P 29; n = 4) (P 29; n =6) Normalized to mg-proteins Complex I 0.05 ± 0.01 0.05 ± 0.03   0.04 ±0.0.03 0.07 ± 0.03 0.24 ± 0.13 0.29 ± 0.06 Complex I + III 0.79 ± 0.1 0.88 ± 0.1  0.60 ± 0.1 0.89 ± 0.4  0.62 ± 0.38 0.71 ± 0.16 Complex II +III 0.36 ± 0.04 0.38 ± 0.03  0.33 ± 0.03 0.30 ± 0.06 0.47 ± 0.05 0.44 ±0.01 Complex IV  0.3 ± 0.1*  0.17 ± 0.05* 0.17 ± 0.1 0.22 ± 0.13 1.98 ±0.29 1.55 ± 0.17 Complex II 0.4 ± 0.1 0.51 ± 0.1  0.27 ± 0.1 0.43 ± 0.060.98 ± 0.18 0.79 ± 0.04 CS  5.3 ± 1**  7.5 ± 1** 6.05 ± 0.8 7.2 ± 1  7.2± 1.2 7.09 ± 0.9  Normalized to CS Complex I 0.009 ± 0.003 0.007 ± 0.003 0.007 ± 0.004  0.01 ± 0.005  0.03 ± 0.022 0.042 ± 0.009 Complex I + III0.15 ± 0.05 0.11 ± 0.01  0.1 ± 0.03  0.12 ± 0.056 0.08 ± 0.04  0.1 ±0.02 Complex II + III  0.07 ± 0.014  0.05 ± 0.006*  0.05 ± 0.01 0.04 ±0.01 0.066 ± 0.011 0.062 ± 0.007 Complex IV 0.057 ± 0.03   0.02 ± 0.008*0.028 ± 0.01  0.03 ± 0.017 0.28 ± 0.06 0.22 ± 0.04 Complex II 0.07 ±0.02 0.069 ± 0.01   0.045 ± 0.019  0.06 ± 0.017 0.14 ± 0.03 0.11 ± 0.01Normalized to II Complex I 0.13 ± 0.05 0.11 ± 0.07  0.11 ± 0.04 0.18 ±0.1  0.25 ± 0.12 0.37 ± 0.08 Complex I + III 2.07 ± 0.6  1.75 ± 0.3 2.34 ± 0.5 2.06 ± 0.9  0.74 ± 0.7  0.89 ± 0.15 Complex II + III 0.97 ±0.25 0.76 ± 0.15 1.37 ± 0.4  0.7 ± 0.05 0.50 ± 0.15 0.55 ± 0.02 ComplexIV 0.79 ± 0.3  0.36 ± 0.1  0.66 ± 0.3 0.55 ± 0.3  2.06 ± 0.4  1.96 ±0.13 CS 14.2 ± 4.2  15.01 ± 3.2  19.4 ± 4.6 17.05 ± 4.1  7.78 ± 3   8.9± 1.2 Data expressed in micromole/min/mg tissue and normalized tomg-proteins (mean ± SD) or normalized to citrate synthase (CS) activityor to complex II activity (mean ± SD). Statistical analyses wereperformed with untreated Tk2^(−/−) vs untreated Tk2⁺ (P 13) andTk2^(−/−200dCMP/dTMP) vs Tk2^(+200dCMP/dTMP) (P 13 and P 29). *= p <0.05; **= p < 0.005). P = postnatal day

TABLE 3 Cerebellar mitochondrial respiratory chain enzyme activitiesNormalized to CS COX I II+III II I+III Tk2⁺  0.24 ± 0.07  0.08 ± 0.03 0.11 ± 0.02  0.11 ± 0.03  0.14 ± 0.03 Tk2^(−/−)  0.13 ± 0.07* 0.015 ±0.01**  0.06 ± 0.02*  0.06 ± 0.01* 0.067 ± 0.05* Tk2⁺¹³ ^(days 200) 0.24 ± 0.07 0.065 ± 0.04  0.11 ± 0.03  0.11 ± 0.003  0.11 ± 0.06Tk2^(−/−13 days 200)  0.19 ± 0.01 0.036 ± 0.01  0.10 ± 0.02 0.087 ± 0.06 0.11 ± 0.01 Tk2^(+29 days)  0.20 ± 0.02 0.069 ± 0.007 0.072 ± 0.0040.073 ± 0.008  0.10 ± 0.01 Tk2^(+29 days 200)  0.17 ± 0.03 0.049 ± 0.0040.071 ± 0.02 0.065 ± 0.01  0.11 ± 0.03 Tk2^(−/−29 days 200)  0.11 ±0.03* 0.034 ± 0.01 0.057 ± 0.01 0.080 ± 0.02  0.05 ± 0.005*Tk2^(+29 days) 0.027 ± 0.002 0.002 ± 0.002 0.039 ± 0.009  0.07 ± 0.010.028 ± 0.002 Tk2^(+29 days 400) 0.036 ± 0.022 0.005 ± 0.001 0.017 ±0.012 0.035 ± 0.02 0.027 ± 0.019 Tk2^(−/−29 days 400)  0.04 ± 0.0240.004 ± 0.002  0.02 ± 0.006  0.05 ± 0.01 0.028 ± 0.019 Normalized to IICOX I II+III CS I+III Tk2⁺  2.1 ± 0.6  0.74 ± 0.3  1.01 ± 0.1  9.1 ± 2.5 1.31 ± 0.2 Tk2^(−/−)  2.1 ± 0.7  0.29 ± 0.2  1.06 ± 0.1  18.5 ± 7.4 0.97 ± 0.6 Tk2^(+13 days 200)  2.09 ± 0.2  0.43 ± 0.3  0.98 ± 0.1  9.1± 2.5  0.92 ± 0.3 Tk2^(−/−13 days 200)  1.7 ± 0.4  0.29 ± 0.1  0.84 ±0.0.5  8.8 ± 1.8  0.94 ± 0.1 Tk2^(+29 days)  2.8 ± 0.02  0.95 ± 0.003 0.99 ± 0.1  13.7 ± 1.5  1.48 ± 0.08 Tk2^(+29 days 200)  2.62 ± 0.38 0.65 ± 0.4  0.97 ± 0.1  15.9 ± 3.6  1.76 ± 0.2 Tk2^(−/−29 days 200) 1.43 ± 0.4  0.43 ± 0.2  0.72 ± 0.1  13.3 ± 3.5  0.68 ± 0.2Tk2^(+29 days)  0.38 ± 0.05 0.031 ± 0.04  0.53 ± 0.04   14 ± 2.2  0.4 ±0.03 Tk2^(+29 days 400)  0.43 ± 0.3  0.14 ± 0.02  0.35 ± 0.03   16 ± 4.3 0.56 ± 0.3 Tk2^(−/−29 days 400)  0.47 ± 0.3  0.05 ± 0.06  0.41 ± 0.01 19.4 ± 3.4f  0.5 ± 0.2 Data expressed in micromole/min/mg tissue andnormalized to mg-proteins or normalized to citrate synthase (CS)activity or to complex II activity (mean ± SD). Statistical analyseswere performed with untreated Tk21^(−/−) vs untreated Tk2⁺ andTk2^(−/−200dCMP/dTMP) vs Tk2^(+200dCMP/dTMP). *= p <0.05; * = p <0.005)P = postnatal day

Example 6 Deoxynucleotide Metabolism

To understand the metabolism of dCMP/dTMP after oral gavageadministration, levels of dCMP/dTMP and their metabolites in muscle andliver tissues and in plasma were measured after 30 minutes of oralgavage. In Tk2^(−/−200dCMP/dTMP) mice, deoxynucleoside monophosphateswere not detectable. Levels of deoxyuridine and deoxythymidine weremarkedly increased at age 13 days, but subsequently lower in 29-day-oldmice (FIGS. 8A-8C). Thymidine phosphorylase (TP) degrades deoxyuridineand deoxythymidine, respectively, to uracil and thymine plus deoxyribose1-phosphate (Brown and Bicknell 1998; Hirano, et al. 2004).

To understand the cause for differences in deoxyuridine anddeoxythymidine plasma levels between 13 and 29 days of age, the activitylevel of TP in small intestine, brain, and liver was measured. TPactivity was higher in the small intestine at P29 (FIG. 8D), butunchanged in brain and liver tissues (Table 4). Therefore, intestinal TPis responsible for the rapid catabolism of deoxyuridine anddeoxythymidine at P29 and the resulting reduced plasma levels.

Tk2 activity was confirmed to be normal in treated and untreatedTk2+mice and reduced in Tk2^(−/−) mice in muscle and brain.Unexpectedly, Tk1 activity was increased in brain and muscle of 13- and29-day-old treated mice (FIGS. 8E and 8F).

TABLE 4 Deoxypyrimidine monophosphates metabolism Plasma Levels ofnucleoside metabilites Mice Age Treatment Dose Time Uracil (μM) Thymine(μM) dUrd (μM) Thd (μM) Tk2⁺ (N = 1) 13 dCMP + dTMP 80 nmol/kg/day 019.1 UND 5.5 1.0 Tk2⁺ (N = 3) 13 dCMP + dTMP 80 nmol/kg/day 30 59.3 ±33.9 3.6 ± 3.3 29.0 ± 18.5 71.6 ± 53.3 Tk2^(−/−) (N = 1) 13 dCMP + dTMP80 nmol/kg/day 0 21.2 5.0 4.2 6.8 Tk2^(−/−) (N = 3) 13 dCMP + dTMP 80nmol/kg/day 30 78.0 ± 56.7 5.4 ± 3.5 28.3 ± 9.1  70.4 ± 19.2 Tk2⁺ (N =3) 29 dCMP + dTMP 80 nmol/kg/day 0 79.7 ± 20.5 0.1 ± 0.1 2.2 ± 0.1 5.0 ±5.4 Tk2⁺ (N = 2) 29 dCMP + dTMP 80 nmol/kg/day 30 109.3 ± 1.3  68.7 ±53.3 29.3 ± 13.4 73.4 ± 50.2 Tk2^(−/−) (N = 3) 29 dCMP + dTMP 80nmol/kg/day 30 61.4 ± 10.9 11.2 ± 3.4   18 ± 1.9 30.8 ± 13.2 ThymidinePhosphorylase activity Mice Age Treatment Dose TISSUE Activity(nmol/h/mg-proteins) Tk2⁺ (N = 3) 13 dCMP + dTMP 80 nmol/kg/day BRAIN6.2 ± 3.3 Tk2^(−/−) (N = 4) 13 dCMP + dTMP 80 nmol/kg/day BRAIN 11.6 ±0.8  Tk2⁺ (N = 2) 29 dCMP + dTMP 80 nmol/kg/day BRAIN 5.9 ± 0.4Tk2^(−/−) (N = 2) 29 dCMP + dTMP 80 nmol/kg/day BRAIN 8.0 ± 0.8 Tk2⁺ (N= 4) 13 Untreated — LIVER 4.9 ± 2.4 Tk2^(−/−) (N = 2) 13 Untreated —LIVER 6.1 ± 2.6 Tk2⁺ (N = 3) 13 dCMP + dTMP 80 nmol/kg/day LIVER 9.0 ±2.9 Tk2^(−/−) (N = 3) 13 dCMP + dTMP 80 nmol/kg/day LIVER 6.0 ± 3.1 Tk2⁺(N = 6) 29 dCMP + dTMP 80 nmol/kg/day LIVER  4.9 ± 2.45 Tk2^(−/−) (N =4) 29 dCMP + dTMP 80 nmol/kg/day LIVER 4.6 ± 2.5 Tk2⁺ (N = 6) 13Untreated — S.I. 26.1 ± 11.0 Tk2^(−/−) (N = 3) 13 Untreated — S.I. 28 ±14 Tk2⁺ (N = 2) 13 dCMP + dTMP 80 nmol/kg/day S.I. 29.6 ± 3.9  Tk2^(−/−)(N = 3) 13 dCMP + dTMP 80 nmol/kg/day S.I. 30.9 ± 10.6 Tk2⁺ (N = 9) 29dCMP + dTMP 80 nmol/kg/day S.I. 278.8 ± 75   Tk2^(−/−) (N = 4) 29 dCMP +dTMP 80 nmol/kg/day S.I. 244.1 ± 80   S.I. = small intestine; dUrd =deoxyuridine; Thd = thymidine

Example 7 Materials and Protocols Used for Examples 8-10

The compounds used in Examples 8-10 were dTMP.Na₂ and dCMP.Na₂ wereobtained through Hongene Biotechnology USA, Inc. (Morrisville, N.C.).The compounds were verified as greater than 99% pure by HLPC and by massspectrometry. Each bottle of dTMP.Na₂, and dCMP.Na₂ was labeled with thefollowing but not limited to: the product name, product lot number,protocol number, recommended storage conditions, expiration date,“Caution: New Drug-Limited by Federal Law to Investigational Use,”Hongene Biotech' company name and address, and the Investigator's nameor investigation site. Labeling complied with the requirements of 21 CFR312.6.

The Investigator ensured that all study drugs were stored and dispensedin accordance with the Food and Drug Administration (FDA) regulationsconcerning the storage and administration of investigational drugs.

Example 8 dTMP and dCMP Treatment on Patients—Case 1

The first patient was a 29 year-old Spanish man (JJM) with TK2deficiency that initially manifested at age 3 years with leg weaknesscausing difficulty running and frequent falls (Vilà, et at. 2003). Theweakness progressed at age 12, began using BiPAP ventilatory support andone year later, he became wheelchair-bound. Because of arm weakness, hestopped playing the violin and due to respiratory muscle weakness, hehas suffered recurrent bouts of pneumonia and uses BiPAP 23 hours daily.Despite the severe myopathy, this young man completed medical school.

In August, 2011, he began dTMP and dCMP (each 100 mg/kg/day supplied byHongene Biotechnology as described in Example 7), and the dose wasincreased to 200 mg/kg/day. The compounds were mixed in water andadministered orally as a single daily dose. After initiating thistherapy, the patient increased weight from a baseline of 35 kg to 44 kg,muscle strength improved slightly at the biceps, triceps, and quadricepsand he reduced time on BiPAP from 24 to 22 hours daily. During thisperiod, he developed hearing loss, which was likely due to diseaseprogression as this clinical manifestation has been reported in anotherpatient with TK2 deficiency (Marti et al., 2010). No other potentialside-effects were noted by this patient.

Example 9 dTMP and dCMP Treatment on Patients—Case 2

The second patient was a 10 year-old Italian boy (LS DOB Jun. 3, 2004)who presented at age 2 years with hypotonia, severe limb weakness, anddelayed motor development. He was relatively stable and was able to sitand walk unassisted until September, 2011, when he became very weak andlost the ability to walk. He was barely able to stand without support.In March, 2012, he started dTMP+dCMP therapy (dTMP and dCMP (each 100mg/kg/day supplied by Hongene Biotechnology as described in Example 7).The compounds were mixed in water and administered orally as a singledaily dose, which was prescribed by an Italian physician. In August,2012, significant improvement of strength was noted. He was able to walk10 steps independently. In addition to the improved limb strength, hisvoice and head control improved. He grew 5 centimeters in height andgained 3.5 kilograms in weight. In November, 2012, the doses wereincreased to 200 mg/kg/day. In late 2014, his 6-minute walk test showeda 3-fold improvement over pre-treatment baseline. As of March, 2016, heis able to walk unassisted, climb stairs using arm rails for support,ride a bicycle, and swim.

Example 10 dTMP and dCMP Treatment on Patients—Case 3

A third patient was a 3 year-old child (AE, DOB Feb. 11, 2011) withinfantile-onset myopathy. He had documented mt.DNA depletion (11% ofnormal in skeletal muscle), and compound heterozygous mutations in theTK2 gene: c.144_145DelGA mutation which causes a frameshift, andc.323C>T missense mutation (p.T108M) (identified in the Medical GeneticsLaboratory at the Baylor College of Medicine). The child developednormally until age 11 months, when he was able to pull to a standingposition and walk a few steps using his arms to hold onto furniture. Helost the ability to walk and has become unable to sit, stand, roll overin bed, or lift his limbs or head off of a bed. Baseline serum creatinekinase levels were 1290-2098 U/L and venous lactate was elevated at 3.9mmol/I.: (normal 0.5-2.2). The diagnosis of mitochondrial disease wasmade at Johns Hopkins Medical Center in May, 2012, when a muscle biopsyrevealed severe cytochrome c oxidase (complex IV) deficiency. He starteda cocktail of coenzyme Q10, L-carnitine, and creatine, which did notlead to significant improvements. The diagnosis of TK2 deficiency wasachieved by genetic test results in July, 2012. After developing anupper respiratory viral infection on September 2012, he was put onnon-invasive ventilation and subsequently received a tracheostomy andplaced on mechanical ventilation.

An emergency IND was requested on Oct. 24, 2012 to initiate dCMP+dTMPtherapy in AE. At that time, he weighed 10.4 kg and was unable to gripobjects or lift his legs from the bed, An eIND was obtained and hestarted the therapy in November, 2012 at 100 mg/kg/day, One month later,the doses were increased to 200 mg/kg/day. He showed improvement in his,weight, arm strength and leg movements. At age 4 years 9 months, heweighed 19.5 kg and was able to grip and hold small objects, lift hisarm using triceps muscle, and lift his legs from the bed.

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1. A method of treating mitochondrial DNA depletion syndrome in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a composition comprising at leastone deoxyribonucleoside monophosphate.
 2. (canceled)
 3. The method ofclaim 1, wherein the mitochondrial DNA depletion syndrome is chosen fromthe group consisting of deoxyguanosine kinase (dGK) deficiency,thymidine phosphorylase (TP) deficiency, and mutations in the RRM2Bgene.
 4. The method of claim 1, wherein the subject is a mammal.
 5. Themethod of claim 1, wherein the subject is a human.
 6. The method ofclaim 1, wherein the composition comprises two or moredeoxyribonucleoside monophosphates. 7-10. (canceled)
 11. The method ofclaim 1, wherein the therapeutically effective amount is between about100 mg/kg/day and about 1000 mg/kg/day.
 12. The method of claim 1,wherein the therapeutically effective amount is between about 200mg/kg/day and about 800 mg/kg/day.
 13. The method of claim 1, whereinthe therapeutically effective amount is between about 250 mg/kg/day andabout 400 mg/kg/day.
 14. The method of claim 11, wherein the compositioncomprises more than one deoxyribonucleoside monophosphate and thetherapeutically effective amount is between about 100 mg/kg/day andabout 1000 mg/kg/day of each deoxyribonucleoside monophosphate in thecomposition.
 15. The method of claim 11, wherein the compositioncomprises more than one deoxyribonucleoside monophosphate and thetherapeutically effective amount is between about 100 mg/kg/day andabout 1000 mg/kg/day of the total deoxyribonucleoside monophosphates inthe composition.
 16. The method of claim 12, wherein the compositioncomprises more than one deoxyribonucleoside monophosphate and thetherapeutically effective amount is between about 200 mg/kg/day andabout 800 mg/kg/day of each deoxyribonucleoside monophosphate in thecomposition.
 17. The method of claim 12, wherein the compositioncomprises more than one deoxyribonucleoside monophosphate and thetherapeutically effective amount is between about 200 mg/kg/day andabout 800 mg/kg/day of the total deoxyribonucleoside monophosphates inthe composition.
 18. The method of claim 13, wherein the compositioncomprises more than one deoxyribonucleoside monophosphate and thetherapeutically effective amount is between about 250 mg/kg/day andabout 400 mg/kg/day of each deoxyribonucleoside monophosphate in thecomposition.
 19. The method of claim 13, wherein the compositioncomprises more than one deoxyribonucleoside monophosphate and thetherapeutically effective amount is between about 250 mg/kg/day andabout 400 mg/kg/day of the total deoxyribonucleoside monophosphates inthe composition.
 20. The method of claim 1, wherein the composition isadministered once daily, twice daily, three times daily, four timesdaily, five times daily or six times daily.
 21. The method of claim 1,wherein the composition administered orally, intrathecally, enterally,or intravenously.
 22. The method of claim 21, wherein the composition isadministered orally and comprises deoxyribonucleoside monophosphatesmixed with cow's milk, human breast milk, infant formula or water.23-26. (canceled)
 27. The method of claim 1, wherein the therapeuticallyeffective amount of the composition administered to the subject isincreased over time.
 28. The method of claim 27, wherein a firsttherapeutically effective amount of the composition administered to thesubject is about 100 mg/kg/day of composition, and wherein thetherapeutically effective amount of the composition is increased overtime to 200 mg/kg/day, to 400 mg/kg/day, to 800 mg/kg/day, up to 1000mg/kg/day. 29-32. (canceled)
 33. The method of claim 2, wherein themitochondrial DNA depletion syndrome is deoxyguanosine kinase (dGK)deficiency and the deoxyribonucleoside monophosphate is chosen from thegroup consisting of deoxyadenosine monophosphate (dAMP), deoxyguanosinemonophosphate (dGMP), and mixtures thereof and mixtures thereof.
 34. Themethod of claim 2, wherein the mitochondrial DNA depletion syndrome isthymidine phosphorylase (TP) deficiency and the deoxyribonucleosidemonophosphate is thymidine 5′-monophosphate (TMP).